Universiẗat, Diss., 2004--Stuttgart.

Techn. Hochsch., Diss., 2005--Aachen.

Doctor of Philosophy / Department of Agronomy / V. P. Vara Prasad / Crop assessment and monitoring is important to crop management both at crop production level and research plot level, such as high-throughput phenotyping in breeding programs. Optical sensors based agricultural applications have been around for decades and have soared over the past ten years because of the potential of some new technologies to be low-cost, accessible, and high resolution for crop remote sensing which can help to improve crop management to maintain producers’ income and diminish environmental degradation. The overall objective of this study was to develop methods and compare the different optical sensors in crop assessment and monitoring at different scales and perspectives. At crop production level, we reviewed the current status of different optical sensors used in precision crop production including satellite-based, manned aerial vehicle (MAV)-based, unmanned aircraft system (UAS)-based, and vehicle-based active or passive optical sensors. These types of sensors were compared thoroughly on their specification, data collection efficiency, data availability, applications and limitation, economics, and adoption. At research plot level, four winter wheat experiments were conducted to compare three optical sensors (a Canon T4i® modified color infrared (CIR) camera, a MicaSense RedEdge® multispectral imager and a Holland Scientific® RapidScan CS-45® hand-held active optical sensor (AOS)) based high-throughput phenotyping for in-season biomass estimation, canopy estimation, and grain yield prediction in winter wheat across eleven Feekes stages from 3 through 11.3. The results showed that the vegetation indices (VIs) derived from the Canon T4i CIR camera and the RedEdge multispectral camera were highly correlated and can equally estimate winter wheat in-season biomass between Feekes 3 and 11.1 with the optimum point at booting stage and can predict grain yield as early as Feekes 7. Compared to passive sensors, the RapidScan AOS was less powerful and less temporally stable for biomass estimation and yield prediction. Precise canopy height maps were generated from a CMOS sensor camera and a multispectral imager although the accuracy could still be improved. Besides, an image processing workflow and a radiometric calibration method were developed for UAS based imagery data as bi-products in this project. At temporal dimension, a wheat phenology model based on weather data and field contextual information was developed to predict the starting date of three key growth stages (Feekes 4, 7, and 9), which are critical for N management. The model could be applied to new data within the state of Kansas to optimize the date for optical sensor (such as UAS) data collection and save random or unnecessary field trips. Sensor data collected at these stages could then be plugged into pre-built biomass estimation models (mentioned in the last paragraph) to estimate the productivity variability within 20% relative error.

Mitochondria exist in a dynamic network whereby fusion and fission events are critical to the health of the mitochondria, the cell, and the organism. Dysfunctional mitochondrial dynamics underlie a plethora of diseases including various cancers, heart diseases, diabetes, neurodegenerative diseases, and a number of mitochondrial disorders. Despite a strong molecular knowledge of a handful of functional mediators of mitochondrial dynamics, much less is known about how this process is regulated at a cellular level, and what genes are involved in signaling pathways. A previously completed mitochondrial morphology genome screen was repeated with an automated confocal microscope resulting in the identification and validation of MTX2 as a novel regulator of mitochondrial dynamics. Functional characterization of the role of MTX2 in mitochondrial dynamics will further our understanding of mitochondrial biology, and has the future potential to inform therapies for some of the many diseases underscored by dysfunctional mitochondrial dynamics.

Concomitant with antibiotic use is the development of bacterial strains that are resistant to such compounds. Presently, the rate at which antibiotic-resistant pathogenic bacteria are emerging is outpacing our resupply of new antibacterials; therefore, renewed efforts to identify novel therapies are urgently needed. Transition metals are required by all life forms and, for bacteria, an adequate supply of nutrient metal is necessary to establish infection in a host. Indeed, as an antibacterial defense mechanism, eukaryotes have developed various means by which to restrict the availability of metal to the invading pathogen, thereby limiting its chances for successful colonization. As such, bacterial metal acquisition and homeostasis have been suggested as potential antibiotic targets to explore for the identification of new antibacterial small molecules. In this thesis I discuss my development of a high-throughput screening assay that specifically selects for compounds that perturb bacterial iron homeostasis. The results of this work led to the identification of a series of spiro-indoline-thiadiazole compounds that are toxic to bacteria via iron chelation. In addition to molecules that perturb the availability of bacterial intracellular iron, we present a series of spiro-indoline-thiadiazole analogs that inhibit bacterial growth by limiting zinc availability. Furthermore, we show that the respective zinc-perturbing analogs re-sensitize an otherwise drug-resistant strain of NDM-1-harbouring Klebsiella pneumoniae to carbapenem antibiotics. We discuss the potential for this class of compounds to serve as carbapenem adjuvants for treating infections caused by metallo-β-lactamase-containing pathogens. / Thesis / Doctor of Philosophy (PhD)

<p> The high-throughput micro-adhesion tester (HMAT) was constructed to test the adhesive strength of polymers. The design criteria included the ability to rapidly test many different samples in a serial format, and a probe design that would compliment this objective by being easy to place and pull from the samples and easy to mass produce. The HMAT was able to perform 48 adhesion tests at about 30s per test for a total of 24 min. The final probes were made from a capillary tube with a small metal cap on the top for ease of lifting. They are easy to make and easy to place and pull from the custom probe box. The probe box was designed to hold the probes upright while the polymer is drying and during the test while not interfering with the test itself. Tests on PDMS show reasonable repeatability with the standard deviation being about 20% of the mean value. Since the HMAT is meant to be used for primary screening, the accuracy of the measurements is not as critical as it would be for later tests. </p> / Thesis / Master of Applied Science (MASc)

With growing concerns about global food supply and environmental impacts of modern agriculture, we are seeing an increased demand for more horticultural research. While research into plant genetics has seen an increased throughput from recent technological advancements, plant phenotypic research throughput has lagged behind. Improvements in open-source image processing software and image capture hardware have created an opportunity for the development of more competitively-priced, faster data-acquisition tools. These tools could be used to collect measurements of plants' phenotype on a much larger scale without sacrificing data quality. This paper demonstrates the feasibility of creating such a tool. The resulting design utilized stereo vision and image processes in the OpenCV project to measure a representative collection of observable plant traits like leaflet length or plant height. After the stereo camera was assembled and calibrated, visual and stereo images of potato plant canopies and tubers(potatoes) were collected. By processing the visual data, the meaningful regions of the image (the canopy, the leaflets, and the tubers) were identified. The same regions in the stereo images were used to determine plant physical geometry, from which the desired plant measurements were extracted. Using this approach, the tool had an average accuracy of 0.15 inches with respect to distance measurements. Additionally, the tool detected vegetation, tubers, and leaves with average Dice indices of 0.98, 0.84, and 0.75 respectively. To compare the tool's utility to that of traditional implements, a study was conducted on a population of 27 potato plants belonging to 9 separate genotypes. Both newly developed and traditional measurement techniques were used to collect measurements of a variety of the plants' characteristics. A multiple linear regression of the plant characteristics on the plants' genetic data showed that the measurements collected by hand were generally better correlated with genetic characteristics than those collected using the developed tool; the average adjusted coefficient of determination for hand-measurements was 0.77, while that of the tool-measurements was 0.66. Though the aggregation of this platform's results is unsatisfactory, this work has demonstrated that such an alternative to traditional data-collection tools is certainly attainable. / Master of Science

In this thesis the processing mechanisms of short non-coding RNAs (ncRNAs) is investigated by using data generated by the current method of high-throughput sequencing (HTS). The recently adapted short RNA-seq protocol allows the sequencing of RNA fragments of microRNA-like length (∼18-28nt). Thus, after mapping the data back to a reference genome, it is possible to not only measure, but also visualize the expression of all ncRNAs that are processed to fragments of this specific length. Short RNA-seq data was used to show that a highly abundant class of small RNAs, called microRNA-offset-RNAs (moRNAs), which was formerly detected in a basal chordate, is also produced from human microRNA precursors. To simplify the search, the blockbuster tool that automatically recognizes blocks of reads to detect specific expression patterns was developed. By using blockbuster, blocks from moRNAs were detected directly next to the miR or miR* blocks and could thus easily be registered in an automated way. When further investigating the short RNA-seq data it was realized that not only microRNAs give rise to short ∼22nt long RNA pieces, but also almost all other classes of ncRNAs, like tRNAs, snoRNAs, snRNAs, rRNAs, Y-RNAs, or vault RNAs. The formed read patterns that arise after mapping these RNAs back to a reference genome seem to reflect the processing of each class and are thus specific for the RNA transcripts of which they are derived from. The potential of this patterns in classification and identification of non-coding RNAs was explored. Using a random forest classifier which was trained on a set of characteristic features of the individual ncRNA classes, it was possible to distinguish three types of ncRNAs, namely microRNAs, tRNAs, and snoRNAs. To make the classification available to the research community, the free web service ‘DARIO’ that allows to study short read data from small RNA-seq experiments was developed. The classification has shown that read patterns are specific for different classes of ncRNAs. To make use of this feature, the tool deepBlockAlign was developed. deepBlockAlign introduces a two-step approach to align read patterns with the aim of quickly identifying RNAs that share similar processing footprints. In order to find possible exceptions to the well-known microRNA maturation by Dicer and to identify additional substrates for Dicer processing the small RNA sequencing data of a Dicer knockdown experiment in MCF-7 cells was re-evaluated. There were several Dicer-independent microRNAs, among them the important tumor supressor mir-663a. It is known that many aspects of the RNA maturation leave traces in RNA sequencing data in the form of mismatches from the reference genome. It is possible to recover many well- known modified sites in tRNAs, providing evidence that modified nucleotides are a pervasive phenomenon in these data sets.

This dissertation describes the development of a system for the automated, high-throughput screening of organic reactions. This system utilizes a liquid handling robot for reaction mixture preparation combined with desorption electrospray ionization mass spectrometry (DESI-MS) for reaction mixture analysis. With an analysis speed of ~1 second per reaction mixture, this system is capable of screening thousands of reactions per hour. Reaction mixtures are prepared in 384-well microtiter plates using a liquid handling robot. A sample of each reaction mixture (50 nL) is then transferred to a PTFE coated, glass slide using a pin tool. By offsetting the placement of the pin tool during each transfer, up to 6,144 unique reaction mixtures can be placed on each slide. The slide is then transferred to the DESI stage by a robotic arm, and the DESI-MS analysis begins, taking as little as 7 minutes for 384 reaction mixtures. We utilize a scheduling software to control each component of the system, which automates the entire process from reaction mixture preparation to DESI-MS analysis. In order to efficiently analyze and visualize the extremely large data sets generated by the system, we developed a custom software suite to automatically process each data set. We have used this system to screen several classes of industrially relevant reactions including Suzuki coupling, nucleophilic aromatic substitution, reductive amination, and Sonogashira coupling. We have validated both positive and negative results from the system using flow chemistry, and we have observed excellent agreement between the two methodologies. By being capable of screening thousands of reactions per hour, requiring only microliter quantities of reaction mixtures, and consuming less than a milliliter of solvent during the DESI-MS analysis, this system significantly reduces the time and costs associated with organic reaction screening.

La microfluidique diphasique permet la production et la manipulation de millions de gouttelettes hautement monodisperses, chacune d'entre elles peut servir de microréacteur indépendant. Cette technologie offre de grandes perspectives dans de nombreux domaines scientifiques et industriels (principalement en biotechnologie). Les gouttelettes peuvent être produites, analysées et manipulées à très haut débit grâce notamment à des méthodes optiques. De nombreuses études ont été menées pour améliorer cette technologie et ses applications mais une compréhension approfondie des processus dynamiques complexes se produisant à l'interface des gouttelettes et du fluide porteur (huile) n'est toujours pas bien comprise. Au cours de cette thèse, nous nous sommes intéressés au développement d’une nouvelle approche optofluidique permettant une meilleure analyse de la dynamique et l'organisation moléculaire aux interfaces des gouttelettes dans l’écoulement microfluidique.Notre première étude porte sur la conception et l'utilisation de surfactants photosensibles permettant de stabiliser les gouttelettes et d’induire ensuite leur fusion contrôlée à l'aide d’un laser UV pulsé. La lumière offrant une grande flexibilité, une accordabilité (longueur d’onde et intensité) et une haute résolution spatio-temporelle. Deux approches ont été développées : l’une basée sur la photolyse de molécules photolabiles (processus irréversible) et l’autre basée sur la photo-isomérisation de dérivées d'azobenzène (processus réversible). Le succès de ces deux approches n’était pas évident, car l’irradiation de l’interface à l’échelle microscopique induit des modifications au niveau de la tension interfaciale mais aussi au niveau des processus de diffusion et d’absorption des molécules tensio-actives à l’interface, chacune de ces étapes ajoute une échelle de temps et une distance caractéristique différente. Nos résultats ont permis de déterminer le coefficient de diffusion des surfactants dans la région de l’interface, ainsi que le véritable mécanisme de fusion des gouttes par photo-isomérisation.La deuxième étude porte sur la détection et l'analyse en temps réel des propriétés optiques de l’interface et ce afin de mieux comprendre sa construction, sa dynamique et l'organisation moléculaire dans l’écoulement hydrodynamique. Nous avons pour cela mis au point un système original de détection hautement sensible à large bande, utilisant un réflecteur parabolique ne nécessitant pas l’utilisation de filtres dichroïques. Nous obtenons ainsi une détection en temps réel ultra-sensible de la photoluminescence des gouttelettes sur une large plage spectrale. Nous avons mis en évidence pour la première fois l’apparition d’une émission anti-stokes, thermiquement activée (hot band emission). Celle-ci est principalement localisée au niveau de l’interface. Notre dispositif pourrait constituer un nouvel outil puissant d’analyse permettant de détecter et d’étudier les interfaces liquides avec une très grande résolution spatiale, temporelle et spectrale sans recourir à des techniques complexes d’imagerie et de microscopie optique. Nous montrons par exemple que, contrairement à la microscopie optique, l’émission anti-stokes mise en évidence permet de détecter la formation de vésicules (émulsion double) en temps réel et à très haut débit.Lors de la dernière étude, nous nous intéressons à la diffusion de colorants à travers des bicouches biomimétiques dans des systèmes microfluidiques. Deux approches ont été abordées, celle des bicouches à l’interface de microgouttes (Droplet Interface Bilayer) et celle des émulsions doubles (eau/huile/eau). Nos résultats préliminaires montrent que de tels systèmes constituent de bons modèles pour l’étude du transport de molécules et de médicaments à travers des membranes biologiques. / Droplet microfluidics offers tremendous applications and prospects in many scientific and industrial fields (mainly in biotechnology). The technology enables for the fabrication and manipulation of millions of highly monodisperse microdroplets, each of which may be regarded as an independent micro-reactor. Droplets may be produced, monitored, and manipulated at kHz rates, using mainly optical (optofluidics) methods. Numerous studies have been conducted to improve the technology but a thorough understanding of the complex fundamental dynamical processes occurring at the droplets interface are still not well understood. During this PhD work, we focused on the development of new optofluidics approaches for a better understanding of the dynamics and the molecular organization at the droplets interface during droplets production and droplets transport in microfluidic channels and chambers (traps).Our first study concerned the design and use of a droplet-stabilizing photoactive surfactant for a controlled merging of droplets using a ps UV-laser. This is particularly attractive approach since light provides flexibility, wavelength/intensity tunability and high temporal/spatial resolutions. We investigated two different methods: photolysis of photolabile molecules (irreversible process) and photo-isomerization of azobenzene derived molecules (reversible process). The success of approach was far from trivial, since illumination at the microscale induces changes not only in the dynamics of the interfacial tension but triggers also changes in diffusion and absorption of surfactant molecules at the droplets interface, each partial step adding a typical time and length-scale. Analysis of the measured merging time (found at the ms time scale) allowed for the determination of the diffusion coefficient of surfactant molecules around the droplet interface. Another important result was the first experimental demonstration of the mechanism of the light-driven merging process using photo-isomerization. It was found to rely on a subtle opto-mechanical process induced by the switching between trans and cis isomers of azobenzene surfactant molecules under illumination.In our second study, we focused on the real-time detection and analysis of the optical properties of dyes at the droplets interfaces, in order to better understand the building, the dynamics and the molecular organization of the droplet interface in the flow. For this aim, we developed an original broadband highly sensitive detection system, using an off-axis full VIS spectrum - collection, reflection and detection scheme. Our setup enables to achieve a real-time detection of droplets photo-luminescence over a large spectral range and at the ms timescale and to show for the first time the occurring of a thermally activated hot band anti-stokes shift emission. The later was found to localize mainly at the droplets interfaces. Based on this original result, we propose that our optofluidic system may serve as a new analysis tool to detect and study soft interfaces without the aid of optical imaging/recording techniques. The observed hot band anti-stokes shift is shown to be suitable for instance to detect and discriminate between flowing droplets and vesicles (or double emulsions) in a real-time and high throughput detection mode.In the last study, we were particularly interested in the study of mass transport and diffusion of dyes across biomimetic bilayers systems. Two major approaches were addressed, the droplet-interface-bilayer (DIB) and solvent evaporated water/oil/water double emulsions. Both techniques required rigorous design and micro-fabrication characterization. Preliminary results show that such systems may lead to the development of smart applications in soft-bio-mimetic membrane’s design, mass transport and drug carriers studies.